Abstract

Most antibodies that broadly neutralize HIV-1 are highly somatically mutated in antibody clonal lineages that persist over time. Here, we describe the analysis of human antibodies induced during an HIV-1 vaccine trial (GSK PRO HIV-002) that used the clade B envelope (Env) gp120 of clone W6.1D (gp120(W6.1D)). Using dual-color antigen-specific sorting, we isolated Env-specific human monoclonal antibodies (MAbs) and studied the clonal persistence of antibodies in the setting of HIV-1 Env vaccination. We found evidence of V(H) somatic mutation induced by the vaccine but only to a modest level (3.8% ± 0.5%; range 0 to 8.2%). Analysis of 34 HIV-1-reactive MAbs recovered over four immunizations revealed evidence of both sequential recruitment of naïve B cells and restimulation of previously recruited memory B cells. These recombinant antibodies recapitulated the anti-HIV-1 activity of participant serum including pseudovirus neutralization and antibody-dependent cell-mediated cytotoxicity (ADCC). One antibody (3491) demonstrated a change in specificity following somatic mutation with binding of the inferred unmutated ancestor to a linear C2 peptide while the mutated antibody reacted only with a conformational epitope in gp120 Env. Thus, gp120(W6.1D) was strongly immunogenic but over four immunizations induced levels of affinity maturation below that of broadly neutralizing MAbs. Improved vaccination strategies will be needed to drive persistent stimulation of antibody clonal lineages to induce affinity maturation that results in highly mutated HIV-1 Env-reactive antibodies.

Plasma antibody activity induced by GSK PRO HIV-002. (A to C) Vaccine-induced reactivity by isotype. Serial serum samples from vaccine recipients were analyzed for vaccine-specific antibody. Binding antibody multiplex assay data are plotted as estimated antibody concentrations for IgG subclasses; asterisks indicate that antibodies at day 0 were below the limit of detection. IgG subclass responses to HIV-1 gp120 appeared at day 42, with IgG1 responses peaking at day 98 (A). Antibody responses to Nef (B) and Tat (C) also appeared at day 42. The overall concentrations of HIV-1-specific antibody responses mirror the relative concentrations present in blood. (D) Neutralization activity of d182 serum. Serum samples were tested for activity against a panel of Env-pseudotyped viruses in the TZM-bl assay. No neutralizing activity was detected at a 1:20 dilution of preimmunization samples. In contrast, serum samples from day 182 showed neutralization of autologous W6.1D-TCLA.7 Env-pseudotyped virus (geometric mean titer, 1:1645) and neutralization against HIV-1 MN.3 (geometric mean titer, 1:448) in all vaccine recipients. Most vaccine recipients (26/30, or 87%) developed neutralizing activity against HIV-1 SF162.LS (geometric mean titer, 1:75) while a minority developed activity against HIV-1 BaL.26 (9/30, or 30% of subjects with neutralizing activity). (E) ADCC activity of serum IgG. Serum IgG from the four subjects (identified by GSK numbers) from whom rMAbs were recovered was tested for ADCC activity as described. No activity was detected in samples prior to immunization. In contrast, ADCC activity was detected in all samples from day 182 with a peak in granzyme B (GzB) activity comparable to activity levels of the recovered rMAbs (Fig. 4).

Increased maturation of recovered antibodies following repeat immunization. (A) HC mutation frequencies of Env-reactive antibodies after two immunizations (day 42) was 1.8% ± 0.6%; antibodies recovered after four immunizations (day 182 and 672) had a mutation frequency of 3.8% ± 0.5% (P = 0.0095; Mann-Whitney U = 40). (B) HC mutation frequencies of antibodies not reactive with HIV-1 gp120 or gp140 in any assay are shown. The number of antibodies shown appears below each column. Mean mutation frequency ± standard error for each time point was as follows: day 0, 7.7% ± 0.6%; day 42, 6.4% ± 0.3%; day 182, 7.7% ± 0.5%; day 672, 4.6% ± 0.4%. Antibodies recovered at day 672 had a lower mean mutation frequency than those recovered at the other three time points (P < 0.001; Kruskal-Wallis statistic, 20.5). (C) Clonal lineage 15.4.31 consisted of two rMAbs, one isolated at day 42 and one at day 182. Antibody 5148 was nearly unmutated (see Fig. S1 in the supplemental material). (D) CDR H3 length of Env-reactive rMAbs shown as a histogram. Median CDR H3 length was 16.5 amino acids; the mean was 17.2 amino acids. (E) Reactivity of clonal lineage 15.4.31 in binding antibody multiplex (Luminex) assay and ELISA, tested at 1 μg/ml. Antibody 3830 had a 4.7% HC mutation frequency and reacted strongly to gp120W6.1D in both assays. Antibody 5148 had a 0% HC mutation frequency, reacted weakly in the multiplex binding assay, and did not react in ELISA. MFI, mean fluorescence intensity. (F) CDR H3 lengths of all recovered rMAbs, regardless of reactivity, shown as a histogram. Median CDR H3 length was 14 amino acids; the mean was 14.7.

ADCC activity of selected rMAbs. A subset of 14 antibodies was tested for ADCC activity; the granzyme B activity is shown for each rMAb. The cutoff of reactivity is 5%, shown by a horizontal line. Seven rMAbs mediated ADCC, recapitulating the serum activity (Fig. 1E).